Novel primate miRNAs coevolved with ancient target genes in germinal zone-specific expression patterns

Abstract

Major nonprimate-primate differences in cortico-genesis include the dimensions, precursor lineages, and developmental timing of the germinal zones (GZs). microRNAs (miRNAs) of laser-dissected GZ compartments and cortical plate (CP) from embryonic E80 macaque visual cortex were deep sequenced. The CP and the GZ including ventricular zone (VZ) and outer and inner subcompartments of the outer subventricular zone (OSVZ) in area 17 displayed unique miRNA profiles. miRNAs present in primate, but absent in rodent, contributed disproportionately to the differential expression between GZ subregions. Prominent among the validated targets of these miRNAs were cell-cycle and neurogenesis regulators. Coevolution between the emergent miRNAs and their targets suggested that novel miRNAs became integrated into ancient gene circuitry to exert additional control over proliferation. We conclude that multiple cell-cycle regulatory events contribute to the emergence of primate-specific cortical features, including the OSVZ, generated enlarged supragranular layers, largely responsible for the increased primate cortex computational abilities.

Figure 1. miRNA collective variation distinguishes embryonic cortical zones

(A) PCA was applied to the miRNA profiles of the CP (CP17, CP18) and the GZ (OSVZ17int, OSVZ17ext, OSVZ18, VZ17, VZ18) from E80 macaque embryos. Two clusters stand out— CP and GZ—that are independent of the cortical area. (B) PCA using a subset of the data, the OSVZ of area 17 and 18, separated the OSVZ17 (int and ext) from OSVZ18. (C) Samples from area 17 reveal further distinctions between VZ and OSVZ fractions. See also Figure S1 and Table S1.

Figure 2. Heat map for differentially expressed miRNAs

(A) DE miRNAs were statistically identified by EdgeR, FDR< 0.05. CP (17 and 18) were compared to all GZ (17 and 18). (B) For area 17 two additional comparisons were done: VZ17 to OSVZ17int and to OSVZ17ext. (C) Comparison of OSVZ17: OSVZ17int vs OSVZ17ext. DE miRNAs between OSVZ17int and OSVZ17ext have a higher percentage of miRNAs reported in primates, but absent in mouse (54%) than DE miRNAs between CP and GZ (34%). See also Figure S2 and Table S2.

Figure 3. RICStrap DE miRNAs and validation

(A) Using the RISCtrap method, DE mRNAs were pulled down for mir-550-3p, mir-3613-5p, mir-1301-3p, mir-1180-3p, mir-1260a-5p, mir-1271-5p. DE mRNAs were determined using the EdgeR package, FDR<0,05. mll1, mll2, kansl1 and dlx1 are targets of miRNAs expressed in primates and laurasiatheria, but not in rodentia. kansl1 mRNA expression was confirmed by ISH (B), and the presence of the MLL2 protein by immunofluorescence (C) on E80 cortex cryosection. Scale bars: 50 μm. See also Figure S3 and Table S3.

Figure 4. DE miRNAs control key points in the cell cycle and in cortical development

(A) Key proteins which regulate neuronal progenitor cell-cycle and neurogenesis were among the proven targets of the DE miRNAs (Black: miRNAs expressed in primate, Laurasiatheria, and Rodentia; Red, miRNAs expressed in primates and Laurasiatheria, but not in Rodentia). (B) Levels of expression of these DE miRNAs are represented on a heat map.